源自iPSC的人脑组织体的一代新型早期神经发育障碍
Summary
Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.
Abstract
The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue.
This gap is now filled through the application of iPSC-derived, 3D brain organoids, “Brains in a dish,” that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro.
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.
Introduction
人类神经发育障碍，如小头畸形，只能研究甚少在动物模型中由于人类大脑有较长的皮质表面，独特的功能，从非人类动物不同。
该方面使得人脑发育一个复杂的过程，不能被充分研究在2D， 体外细胞培养系统。新兴的3D培养技术允许从诱导的多能干细胞（iPS细胞）的组织样类器官的生成。在3D悬浮培养多能干细胞的体外分化允许各种细胞类型的及时和特定区域的方式形成的，从而产生了一个有组织的，分层组织1，2，3。多亏了3D先锋文化技术和揭秘的器官形成的复杂性，从干细胞开始实验室，我们开发大脑产生对类器官描绘人类大脑发育的早期事件和小头畸形体外 1，2，3模型的鲁棒性的方法。值得注意的是，我们适于通过兰开斯特等人开发的原始方法。以产生脑类器官1。这种方法是根据我们的实验要求进行修改。
从加布里埃尔等人的一项研究的目的。是分析大脑发育过程中的神经干细胞维持的细胞和分子机制。为了做到这一点，一种机械的研究是通过分析在从一个小头畸形患者4衍生的三维大脑类器官的神经祖细胞（NPC）进行。这个病人在CPAP，中心体生物发生所需要5保守中心体蛋白进行的突变。一个被广泛接受的低论点是，小头畸形是NPC池的耗尽的结果，这可能是由于无论是细胞死亡或过早分化1，6，7，8，9。
通过分析头畸形脑类器官的心室区（VZS），已显示的NPC的显著数进行不对称细胞分裂，不同于来自健康供体4衍生的脑类器官。 microcephalic脑组织体的广泛微观和生化分析揭示了及时纤毛拆卸4 CPAP意想不到的作用。具体地，突变的CPAP与延迟纤毛拆卸和延迟细胞周期重新条目相关联，从而导致的NPC 4的过早分化。这些结果表明，在小头畸形和日纤毛作用神经和大脑的大小控制在10 EIR参与。
该协议的第一部分是一个三步骤的方法的描述，以产生均匀的脑类器官。正如前面提到的，原来的兰开斯特协议进行调整和修改，以适应我们的宗旨1。首先，人类iPSC在上Engelbreth-Holm的-群（EHS）矩阵定义的无饲养条件下培养。这一步骤避免了馈线依赖性多能干细胞培养物的变体。在这个协议中，神经分化的诱导以形成神经上皮直接从iPSC的开始。通过跳过胚状体（EB）形成步骤中，以更受控和定向的方式的神经分化前进。该方法限制了其他生殖细胞层，例如中胚层和内胚层的自发和无向的形成。通过施加这个协议中，含有神经花环神经球可以在第5天收获为EHS矩阵的嵌入和固定的悬浮培养。用于我们的协议的第三步骤中的类器官培养基补充有dorsomorphin和SB431542。 Dorsomorphin是骨形态发生蛋白（BMP）的小分子抑制剂，和SB431542抑制TGFβ/激活素/ Nodal信号通路。这些因素的组合可以促进神经分化比更有效地单独11，12，13，14视黄酸。
总之，这些修饰使再现的生成脑组织体，并在保持类器官最小变化。重要的是，施加这种方法来鲁棒地产生从患者的iPSC，其携带突变影响中心体和细胞周期动力学的基因microcephalic脑类器官。
此协议的第二部分发出指令，以制备BR艾因类器官的分析和畸形细胞缺陷的解释。这包括固定，冷冻切片，免疫荧光染色和共聚焦显微镜分析。该协议会为读者提供了预期结果的详细说明和解释为指导。
Protocol
Representative Results
Discussion
MCPH是一个复杂的人类神经发育障碍，不能在动物模型中在体内或在简单的人细胞培养物在体外方法中重现。 MCPH的临床表现开始在第一孕期出现，早期的神经开始时。因此，3D脑组织体代表了可靠的实验系统，MCPH发展模式。此外，三维人体脑组织体是一种理想的方法，因为i）它们允许与各种遗传背景的患者样本的光谱的适配，ⅱ）它们显示包含不同的神经细胞类型组织的组织，并?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由弗里茨·蒂森基金会（Az.10.14.2.152）的支持。我们感谢组织包埋设施和CMMC的显微镜核心设施。我们是由实验室中心体和细胞骨架生物学的成员提供的讨论和技术支持表示感谢。我们感谢李明Gooi校对稿件。
Materials
| Anti-mouse 488 | Invitrogen | A-11001 | Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 |
| Anti-rabbit 647 | Invitrogen | A-21245 | Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 |
| Arl13b | proteintech | 17711-1-AP | ARL13B rabbit polyclonal antibody |
| CELLSPIN system | IBS Integra Bioscience | 183001 | |
| DAPI | Sigma-Aldrich, US | 32670 | 4′,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers |
| DMEM/F-12 | Gibco, US | 31331093 | Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 |
| Dorsomorphin | Sigma-Aldrich, US | P5499 | Compound C; multiple suppliers |
| Embedding medium | AppliChem | A9011, 0100 | Mowiol; embedding medium; multiple suppliers |
| Engelbreth-Holm-Swarm (EHS) matrix | Corning | 354277 | Matrigel hESC-qualified matrix; important: hESC qualified |
| Fish gelatin | Sigma-Aldrich, US | G7765-250ML | Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4°C |
| Glycine | AppliChem | A1067,1000 | Glycine for molecular biology; multiple suppliers |
| Inoculation loop with needle, disposable (1 µl) | Sigma Aldrich, US | BR452201-1000EA | multiple suppliers |
| Insulin | Sigma-Aldrich, US | I3536-100MG | multiple suppliers |
| L-glutamine | Gibco, US | 25030081 | L-glutamine (200 mM) |
| Medium A | Stem cell technologies | #05850 | mTeSR1 (hiPSC medium) |
| Medium B | Stem cell technologies | #05835 | Neural induction medium (NIM); neural differentiation medium |
| Medium C | Gibco, US | 21103049 | Neural Basal Medium |
| MEM | Gibco, US | 11140035 | MEM non-essential amino acids solution (100x) |
| MycoAlert Mycoplasma Detection Kit | Lonza, Switzerland | #LT07-218 | Mycoplasma detection kit; multiple suppliers |
| Nestin | Novus biologicals | NBP1-92717 | Nestin mouse monoclonal antibody (4D11) |
| Paraformaldehyde (PFA) | AppliChem | A3813, 0500 | 4% in PBS, store solution at -20°C; caution: wear skin and eye protection and work under hood |
| PBS tablets | Gibco, US | 18912014 | See manufacturer´s instructions; multiple suppliers |
| Penicillin-Streptomycin (10.000 U/ml) | Gibco, US | 15140122 | Multiple suppliers |
| Poly-L-lysine solution (PLL) | Sigma-Aldrich, US | P8920-100ML | Multiple suppliers |
| pVim | MBL | D076-3S | Phospho-Vimentin (Ser55) mAb |
| Reagent A | Stem cell technologies | # 05872 | Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer´s protocol; alternative products from muliple suppliers available |
| Reagent B | Sigma-Aldrich, US | A6964-100ML | Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 "enzymatic cell dissociation solution” |
| Research Cryostat Leica CM3050 S | Leica biosystems | CM3050 S | Multiple suppliers |
| SB431542 | Selleckchem.com | S1067 | Multiple suppliers |
| Spinner flask 250 ml | IBS Integra Bioscience | 182026 | |
| Sucrose | AppliChem | A4734, 1000 | Multiple suppliers |
| Superfrost ultra plus microscope slides | Thermo scientific, US | J3800AMNZ | Slides should be labeled with a "+" and positively charged |
| Supplement 1 | Gibco, US | 17502048 | N-2 supplement (100x) |
| Supplement 2 w/o Vitamin A | Gibco, US | 12587010 | B-27 supplement (50x), minus vitamin A; multiple suppliers |
| Tissue-Tek Cryomold | Sakura, NL | 4565 | Multiple suppliers |
| Tissue-Tek O.C.T. compound | Sakura, NL | 4583 | Multiple suppliers |
| Triton X-100 | AppliChem | A1388,0500 | Multiple suppliers multiple suppliers |
| TUJ1 | Sigma-Aldrich, US | T2200 | β-Tubulin III (rabbit polyclonal) |
| TUNEL assay | Promega, US | G3250 | DeadEnd Fluorometric TUNEL system; multiple suppliers |
| Tween 20 for molecular biology | AppliChem | A4974,0500 | Multiple suppliers |
| waterproof sheet | BEMIS company, inc. | PM996 | Parafilm “M”; multiple suppliers |
| Y-27632 | Selleckchem.com | S1049 | ROCK-inhibitor (Y-27632 2HCL); multiple suppliers |
| β-mercaptoethanol | Gibco, US | 31350010 | 2-mercaptoethanol (50 mM); multiple suppliers |
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